Studies of the 5' exonuclease and endonuclease activities of CPSF-73 in histone pre-mRNA processing.
Processing of histone pre-mRNA requires a single 3' endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2'-O-methyl nucleotide and occurs in the 5'-to-3' direction. The U7-dependent 5' exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5'-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5' exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5' exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.
Yang
Xiao-cui
XC
Program in Molecular Biology and Biotechnology, CB #3280, University of North Carolina, Chapel Hill, NC 27599, USA.
Sullivan
Kelly D
KD
Marzluff
William F
WF
Dominski
Zbigniew
Z
D000818:Animals; D001483:Base Sequence; D039223:Cleavage And Polyadenylation Specificity Factor / Q000378:metabolism*; D004330:Drosophila; D004720:Endonucleases / Q000378:metabolism*; D005095:Exoribonucleases / Q000378:metabolism*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D020885:Ribonucleoprotein, U7 Small Nuclear / Q000378:metabolism; D013379:Substrate Specificity
D039223:Cleavage And Polyadenylation Specificity Factor; D006657:Histones; D009711:Nucleotides; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D012322:RNA Precursors; D020885:Ribonucleoprotein, U7 Small Nuclear; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D004720:Endonucleases; D005095:Exoribonucleases; C120903:XRN2 protein, human